Sake (Japanese rice wine) production is a complex, multistage process in which fermentation is performed by a succession of mixed fungi and bacteria. This study employed high-throughput rRNA marker gene sequencing, quantitative PCR, and terminal restriction fragment length polymorphism to characterize the bacterial and fungal communities of spontaneous sake production from koji to product as well as brewery equipment surfaces. Results demonstrate a dynamic microbial succession, with koji and early moto fermentations dominated by Bacillus, Staphylococcus, and Aspergillus flavus var. oryzae, succeeded by Lactobacillus spp. and Saccharomyces cerevisiae later in the fermentations. The microbiota driving these fermentations were also prevalent in the production environment, illustrating the reservoirs and routes for microbial contact in this traditional food fermentation. Interrogating the microbial consortia of production environments in parallel with food products is a valuable approach for understanding the complete ecology of food production systems and can be applied to any food system, leading to enlightened perspectives for process control and food safety.
Humans have employed food fermentation since time immemorial to improve the safety, stability, flavor, nutrition, and value of their agricultural products. Traditionally, these processes have been driven by indigenous fungi and bacteria originating in raw materials, in autochthonous starter cultures, or in the processing environment itself (1), organisms that are responsible for these beneficial transformative processes as well as for product spoilage (2, 3). While most modern fermented foods are inoculated with defined starter cultures, traditional, uninoculated products remain celebrated for their historical and cultural significances (4), and indigenous microbial activity is often considered to increase the flavor complexity of these foods (5). The advent of high-throughput sequencing technologies has enhanced our ability to investigate the role of microbial communities in food systems with greater scale and sensitivity than ever possible (4), connecting the transmission of microbial communities in food production and food processing environments to their impact on food products.Sake is the traditional, national alcoholic beverage of Japan. Sake is produced from rice through the saccharification of starch by Aspergillus flavus var. oryzae and subsequent alcoholic fermentation by Saccharomyces cerevisiae. Sake brewing involves a serial propagation process, beginning with koji, a solid culture consisting of rice and A. flavus var. oryzae (6) (Fig. 1). Polished, steamed rice is mixed with the dried spores of A. flavus var. oryzae and incubated for approximately 2 days. Koji is then pitched with more steamed rice, water, and yeast into the moto (seed mash) tank, an open mashing vessel, wherein fermentation occurs for 10 to 25 days. Next, the moto is moved to a larger vessel and mixed with increasing amounts of water, rice, and koji in three additions to form ...